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LacZ‐Based Detection of Acyl‐Homoserine Lactone Quorum‐Sensing Signals

Adam C. Joelsson1,  Jun Zhu1

1University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania

Publication Name: 
Current Protocols in Microbiology
Unit Number: 
UNIT 1C.2
DOI: 
10.1002/9780471729259.mc01c02s3
Online Posting Date: 
December, 2006
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Abstract

Many bacteria use quorum-sensing regulatory systems to monitor their population density and to coordinate their genetic expression under certain conditions. Acyl-homoserine lactones are often produced by many Gram-negative bacteria and serve as quorum-sensing signal molecules. This unit describes two commonly used methods to detect acyl-homoserine lactones from laboratory or environmental bacterial cultures.

Keywords: quorum sensing; acyl-homoserine lactone; LacZ reporter; Agrobacterium tumefaciens

     
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Table of Contents

  • Unit Introduction
  • Basic Protocol 1: Detection of Acyl-Homoserine Lactone Activity Using a Bioassay Strain
  • Basic Protocol 2: Detecting Acyl-Homoserine Lactone Molecules by Thin-Layer Chromatography
  • Support Protocol: Preparation of Preinduced AHL Bioassay Strain
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
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Materials

Basic Protocol 1: Detection of Acyl-Homoserine Lactone Activity Using a Bioassay Strain

 Materials
  • Bacteria of interest
  • Medium appropriate to organism under study (e.g,. see appendix 2C)
  • Preinduced cells (Support Protocol)
  • AT medium (see recipe) without antibiotics
  • Synthetic autoinducer
  • Z buffer (see recipe)
  • 0.05% (w/v) SDS solution in H2O
  • Chloroform
  • 4 mg/ml O-nitrophenyl--d-galactoside (ONPG) in H2O
  • 1 M Na2CO3
  • 10-ml syringe and 0.2-µm syringe filter

Basic Protocol 2: Detecting Acyl-Homoserine Lactone Molecules by Thin-Layer Chromatography

 Materials
  • Bacteria of interest
  • Medium appropriate to organism under study (e.g,. see appendix 2C)
  • Ethyl acetate
  • 60:40 (v/v) methanol/water
  • Preinduced cells (Support Protocol)
  • 2× AT buffer (1:10 dilution of 20× AT buffer; see recipe)
  • 1.5% water-agar (see recipe), 45°C
  • 20 mg/ml 5-bromo-4-chloro-3-indolyl -d-galactopyranoside (Xgal) in dimethylformamide (DMF)
  • 10-ml syringe and 0.2-µm syringe filter
  • 2-ml glass vial
  • TLC tank (30-cm diameter × 10-cm width × 27-cm height)
  • C18 reversed-phase TLC plates (200-mm layer; Baker or Whatman)
  • 28° and 45°C water bath
  • Tape (to seal TLC plates)
  • Sealable plastic container to accommodate TLC plate
  • Additional reagents and equipment for thin-layer chromatography (TLC; Meyers, 2001)

Support Protocol: Preparation of Preinduced AHL Bioassay Strain

 Materials
  • AHL bioassay strain Agrobacterium tumefaciens KYC55 (pJZ372)(pJZ384)(pJZ410); see Zhu (2003)
  • LB medium (appendix 4A) containing 1 µg/ml tetracycline, 100 µg/ml spectinomycin, and
  • 100 µg/ml gentamycin
  • AT medium (see recipe) with and without 1 µg/ml tetracycline, 100 µg/ml spectinomycin, and 100 µg/ml gentamycin
  • 15% (v/v) glycerol, sterile
  • 28°C orbital shaker
  • 2-ml microcentrifuge tubes
Looking for materials?  Suppliers Guide
     
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Figures

  • Figure 1C.2.1
    An example of applying KYC55 (pJZ372)(pJZ384)(pJZ410) to detect AHL-like molecules from the spent medium of a specified Gram-negative bacterium using the liquid assay (A) and the TLC assay (B).

    View Image

Literature Cited

Literature Cited
    Brelles-Mariño, G. and Bedmar, E.J. 2001. Detection, purification and characterisation of quorum-sensing signal molecules in plant-associated bacteria. J. Biotechnol. 91:197-209.
    Dunny, G.M. and Winans, S.C. 1999. Cell-Cell Signaling in Bacteria. ASM Press Washington, D.C.
    Fuqua, W.C., Winans, S.C., and Greenberg, E.P. 1994. Quorum sensing in bacteria: The LuxR-LuxI family of cell density-responsive transcriptional regulators. J. Bacteriol. 176:269-275.
    Fuqua, C., Parsek, M.R., and Greenberg, E.P. 2001. Regulation of gene expression by cell-to-cell communication: Acyl-homoserine lactone quorum sensing. Annu. Rev. Genet. 35:439-468.
    Meyers, C.L.F. 2001. Thin-layer chromatography. In Current Protocols in Nucleic Acid Chemistry (S.L. Beaucage, D.E. Bergstrom, G.D. Glick, and R.A. Jones, eds.) pp. A.3D.1-A.3D.8. John Wiley & Sons, Hoboken, N.J.
    Miller, J.H. 1972. Experiments in Molecular Genetics. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.
    Miller, M.B. and Bassler, B.L. 2001. Quorum sensing in bacteria. Annu. Rev. Microbiol. 55:165-199.
    Shaw, P.D., Ping, G., Daly, S.L., Cha, C., Cronan, J.E. Jr., Rinehart, K.L., and Farrand, S.K. 1997. Detecting and characterizing N-acyl-homoserine lactone signal molecules by thin-layer chromatography. Proc. Natl. Acad. Sci. U.S.A. 94:6036-6041.
    Raleigh, E.A., Elbing, K., and Brent, R. 2002. Selected Topics from Classical Bacterial Genetics. In Current Protocols in Molecular Biology (F.M. Ausubel, R. Brent, R.E. Kingston, D.D. Moore, J.G. Seidman, J.A. Smith, and K. Struhl, eds.) pp. 1.4.1-1.4.14. John Wiley & Sons, Hoboken, N.J.
    Whitehead, N.A., Barnard, A.M., Slater, H., Simpson, N.J., and Salmond, G.P., 2001. Quorum-sensing in Gram-negative bacteria. FEMS Microbiol. Rev. 25:365-404.
    Williamson, L.L., Borlee, B.R., Schloss, P.D., Guan, C., Allen, H.K., and Handelsman J. 2005. Intracellular screen to identify metagenomic clones that induce or inhibit a quorum-sensing biosensor. Appl. Environ. Microbiol. 71:6335-6344.
    Zheng, H., Zhong, Z., Lai, X., Chen, W.X., Li, S., and Zhu, J. 2006. A LuxR/LuxI-type quorum-sensing system in a plant bacterium, Mesorhizobium tianshanense, controls symbiotic nodulation. J. Bacteriol. 188:1943-1949.
    Zhu, J., Beaber, J.W., More, M.I., Fuqua, C., Eberhard, A., and Winans, S.C., 1998. Analogs of the autoinducer 3-oxooctanoyl-homoserine lactone strongly inhibit activity of the TraR protein of Agrobacterium tumefaciens. J. Bacteriol. 180:5398-5405.
    Zhu, J., Chai, Y., Zhong, Z., Li, S., and Winans, S.C. 2003. Agrobacterium bioassay strain for ultrasensitive detection of N-acylhomoserine lactone-type quorum-sensing molecules: Detection of autoinducers in Mesorhizobium huakuii. Appl. Environ. Microbiol. 69:6949-6953.
     
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